11342 • The Journal of Neuroscience, November 1, 2006 • 26(44):11342–11346
Brief Communications
Enhancing GABAA Receptor ␣1 Subunit Levels in
Hippocampal Dentate Gyrus Inhibits Epilepsy Development
in an Animal Model of Temporal Lobe Epilepsy
YogendraSinh H. Raol,1 Ingrid V. Lund,2 Sabita Bandyopadhyay,6 Guojun Zhang,1 Daniel S. Roberts,6,7
John H. Wolfe,1,2,3,5 Shelley J. Russek,6 and Amy R. Brooks-Kayal1,2,4,5
1Division of Neurology, Pediatric Regional Epilepsy Program, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, 2Neuroscience
Graduate Group, 3Center for Comparative Medical Genetics, School of Veterinary Medicine, and Departments of 4Neurology and 5Pediatrics, School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and 6Laboratory of Molecular Neurobiology, Department of Pharmacology and
Experimental Therapeutics, and 7Program in Biomedical Neurosciences, Boston University School of Medicine, Boston, Massachusetts 02118
Differential expression of GABAA receptor (GABR) subunits has been demonstrated in hippocampus from patients and animals with
temporal lobe epilepsy (TLE), but whether these changes are important for epileptogenesis remains unknown. Previous studies in the
adult rat pilocarpine model of TLE found reduced expression of GABR ␣1 subunits and increased expression of ␣4 subunits in dentate
gyrus (DG) of epileptic rats compared with controls. To investigate whether this altered subunit expression is a critical determinant of
spontaneous seizure development, we used adeno-associated virus type 2 containing the ␣4 subunit gene (GABRA4) promoter to drive
transgene expression in DG after status epilepticus (SE). This novel use of a condition-dependent promoter upregulated after SE successfully increased expression of GABR ␣1 subunit mRNA and protein in DG at 1–2 weeks after SE. Enhanced ␣1 expression in DG resulted
in a threefold increase in mean seizure-free time after SE and a 60% decrease in the number of rats developing epilepsy (recurrent
spontaneous seizures) in the first 4 weeks after SE. These findings provide the first direct evidence that altering GABR subunit expression
can affect the development of epilepsy and suggest that ␣1 subunit levels are important determinants of inhibitory function in
hippocampus.
Key words: spontaneous seizures; epilepsy; gene therapy; rat; AAV; GABA
Introduction
GABA is the major inhibitory neurotransmitter in the brain, and
its fast inhibitory action is mediated via GABAA receptors
(GABRs) that are composed of the products (␣1–␣6, ␤1–␤4,
␥1–␥3, ␦, ␧, ␾, ␲) of multiple subunit genes (Macdonald and
Olsen, 1994). Subunit expression and subsequent receptor composition and function vary regionally, developmentally and in
certain disease states. Our previous studies in humans with temporal lobe epilepsy (TLE) and in rodent models of TLE found
reduced expression of GABR ␣1 subunits and increased expression of GABR ␣4 subunits in the dentate gyrus (DG) of epileptic
individuals (Brooks-Kayal et al., 1998, 1999). These changes beReceived Aug. 2, 2006; revised Sept. 7, 2006; accepted Sept. 25, 2006.
This work was supported by National Institutes of Health Grants NS38595 (A.R.B.-K.), NS051710 (A.R.B.-K.,
S.J.R.), NS050393 (S.J.R., A.R.B.-K.), NS51943 (I.V.L.), NS38690 (J.H.W.), DK47757 (J.H.W.), and DK63973 (J.H.W.)
and a grant from the American Epilepsy Society (A.R.B.K., S.J.R.). Funding for statistical support was provided by the
Mental Retardation and Developmental Disability Research Center of the Children’s Hospital of Philadelphia (Grant
P30 HD26979). We are very appreciative for the statistical expertise of Dr. Paul Gallagher in the Division of Biostatistics, Department of Pediatrics at Children’s Hospital of Philadelphia.
Correspondence should be addressed to either of the following: Dr. Amy R. Brooks-Kayal, Children’s Hospital of
Philadelphia, Division of Neurology, Abramson Pediatric Research Center, Room 502, 3615 Civic Center Boulevard,
Philadelphia, PA 19104, E-mail: [email protected]; or Dr. Shelley J. Russek, Laboratory of Molecular Neurobiology, Department of Pharmacology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118,
E-mail: [email protected].
DOI:10.1523/JNEUROSCI.3329-06.2006
Copyright © 2006 Society for Neuroscience 0270-6474/06/2611342-05$15.00/0
gin within 24 h of pilocarpine-induced status epilepticus (SE) in
adult rats and persist for months as these animals uniformly become epileptic (Brooks-Kayal et al., 1998). These subunit alterations are associated with marked changes in receptor function
and pharmacology, including enhanced inhibition of GABA currents by zinc and diminished augmentation of GABA currents by
type I benzodiazepine agonists (Buhl et al., 1996; Gibbs et al.,
1997; Brooks-Kayal et al., 1998). In contrast, rats that experience
pilocarpine-induced SE at postnatal day 10 have increased GABR
␣1 expression in DG and enhanced type I benzodiazepine augmentation of GABA currents, and none develop epilepsy later in
life (Zhang et al., 2004). Together, these findings suggest that
diminished ␣1 levels in dentate granule neurons may contribute
to epileptogenesis and/or that elevated ␣1 levels may be protective, but they do not establish a causal link between changes in
this subunit and epilepsy. To further investigate the potential
importance of GABR ␣1 in TLE, in the present study, we test the
hypothesis that enhancing ␣1 levels in DG after SE using viral
vector gene transfer can inhibit subsequent development of epilepsy in the rat pilocarpine model of TLE.
Materials and Methods
Production of viral vectors. Adeno-associated virus (AAV) vectors were
constructed from pTR–UF4 (Passini et al., 2004) and internal ribosomal
entry site (IRES)– enhanced yellow fluorescent protein (eYFP) (Clon-
Raol et al. • Enhancing ␣1 Subunit Inhibits Epilepsy
tech, Mountain View, CA). The human minimal GABRA4 promoter
(500 bp) (Roberts et al., 2005), with or without the rat ␣1 subunit gene
(GABRA1) cDNA, was directionally cloned into IRES– eYFP. These constructs were then cloned into the pTR–UF4 AAV vector backbone to
create the AAV–␣1 and AAV– eYFP constructs. The recombinant vector
plasmids were packaged into virions by cotransfection of HEK 293T cells
with a helper plasmid and a plasmid expressing the AAV5 serotype capsid. Transfections, purification, and titering were performed by the Penn
Vector Core (University of Pennsylvania, Philadelphia, PA). The
AAV–␣1 vector virus had a titer of 1.9 ⫻ 10 13 genome particles/␮l and
the AAV– eYFP vector virus had a titer of 5.3 ⫻ 10 12 genome particles/␮l.
Viral injection. Adult male Sprague Dawley rats aged 75–120 d
(Charles River Laboratories, Wilmington, MA) were anesthetized with
100 mg/kg ketamine, placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA), and put under continuous halothane (Sigma, St.
Louis, MO) for the duration of the procedure. Two microliters of virus at
a rate of 0.25 ␮l/min was stereotaxically injected into dentate gyrus in
three sites at two different depths/site as described previously (Roberts et
al., 2005).
Status epilepticus induction. At 2 weeks after AAV or sham injections,
rats received intraperitoneal injections of pilocarpine (385 mg/kg) to
induce SE according to a standard protocol (Brooks-Kayal et al., 1998;
Shumate et al., 1998). Rats that did not exhibit convulsive seizures within
1 h of pilocarpine injection were given a second dose of pilocarpine
(192.5 mg/kg) to produce equivalence in seizures between animals. All
rats included in the study developed status epilepticus. Diazepam (6
mg/kg; Hospira, Lake Forest, IL) was administered at 1 h after the onset
of SE to stop seizure activity and was administered again every 2 h (3
mg/kg) until rats stopped seizing.
Behavioral and electroencephalographic monitoring for spontaneous seizures. After completing the viral injection, a subset of rats (n ⫽ 4 for sham
controls and n ⫽ 7 for AAV–␣1) were implanted with electrodes in
frontal cortex and CA1 of hippocampus for combined intracranial electroencephalography (EEG) and video monitoring. All other rats were
assessed using video monitoring only. All rats were monitored continuously starting immediately after pilocarpine injection until the day they
were killed.
RNA amplification and reverse Northern blotting. Dissection of DG and
RNA isolation were performed using standard procedures as described
previously (Roberts et al., 2005; Raol et al., 2006). T7-(dT)24 primer and
SuperScript II RNase H⫺ reverse transcriptase were used for cDNA synthesis, and the AmpliScribe T7 High Yield Transcription kit (Epicenter
Technologies, Madison, WI) was used for RNA amplification according
to published methods (Raol et al., 2006). The cRNA was purified using
the RNeasy MinElute Cleanup kit protocol (Qiagen, Valencia, CA) according to the protocol of the manufacturer. [P 32]dCTP was incorporated into the radiolabeled cDNA probe using SuperScript II RNase H⫺
reverse transcriptase and hybridized for 18 h against a slot blot containing GABR cDNAs (␣1–␣6, ␤1–␤3, ␥1–␥3, ␦, ␧, ␾, ␲) using published
protocols and conditions (Raol et al., 2006).
Reverse transcription-PCR. Reverse transcription-PCR was done using
standard procedures as described previously (Roberts et al., 2005). Each
sample was run in triplicate and contained the following: 1.25 ␮l of
GABR ␣1 Taqman primer probe (Rn00788315_m1; Applied Biosystems,
Foster City, CA) or 1.25 ␮l of Taqman cyclophilin probe (CCG TGT TCT
TCG ACA TCA CGG CTG; Applied Biosystems) with 1.25 ␮l of each
cyclophilin primer (cyclophilin reverse, 5⬘ CCC AAG GGC TCG CCA 3⬘;
cyclophilin forward, 5⬘ TGC AGA CAT GGT CAA CCC C 3⬘; IDT Technologies, Coralville, IA), 12.5 ␮l of Taqman Master mix, and 10 ␮l of
sample cDNA. All values were normalized to cyclophilin expression to
control for loading amount variability.
Protein analysis. Western blotting was performed using our published
protocol (Raol et al., 2006). A total of 12.5 ␮g of DG protein homogenates were run on 10% SDS/polyacrylamide gels and then transferred to
nitrocellulose. Blots were blocked with 5% milk, incubated with 1:400
anti ␣1-subunit primary antibody (Upstate Biotechnology, Lake Placid,
NY) overnight at 4°C, and then incubated with anti-rabbit IgG secondary
antibody conjugated with horseradish peroxidase (HRP) (1:5000 dilution; Amersham Biosciences, Buckinghamshire, UK) using standard
J. Neurosci., November 1, 2006 • 26(44):11342–11346 • 11343
methods. Protein bands were visualized using Super Signal West Pico
chemiluminescent substrate kit (Pierce, Rockford, IL) and quantified
using NIH Image software (National Institutes of Health, Bethesda,
MD). All of the blots were normalized to ␤-actin for which the blots were
striped and reprobed with anti-␤-actin polyclonal antibody at 1:10000
dilution (Sigma) and anti-rabbit IgG secondary antibody conjugated
with HRP, and results are percentage of control values.
Statistical analysis. Because there was no statistical difference between
the control groups (AAV– eYFP, saline, or no injection) killed at the 1, 2,
or 4 week time points after SE in any of the parameters studied, the data
from all control groups were pooled and are represented as “sham control” for statistical comparison with the AAV–␣1-injected rats. Statistical
analysis for the results of reverse Northern, RT-PCR, and Western blotting was done using one-way ANOVA, followed by Bonferroni’s post hoc
test comparing selected groups. Kaplan–Meier survival analysis procedures were used to examine differences between the AAV–␣1 and sham
control rats in time (day) to first spontaneous seizure. Kaplan–Meier
survival analysis is a method for modeling time-to-event data in the
presence of censored cases. The Kaplan–Meier model is based on estimating conditional probabilities at each time point when an event occurs
and taking the product limit of those probabilities to estimate the survival
rate at each point in time. Unpaired Student’s t test was used to analyze
the difference in SE characteristics between AAV–␣1 and sham control
rats. One-way ANOVA, Bonferroni’s, and t test analysis was performed
using Instat (GraphPad Software, San Diego, CA). Kaplan–Meier survival analysis was performed using SAS software, version 8 (SAS Institute,
Cary, NC). A p value ⱕ0.05 was considered statistically significant. Statistical analysis was performed in consultation with the Division of Biostatistics at the Children’s Hospital of Philadelphia.
Results
To directly test the hypothesis that higher ␣1 levels inhibit development of epilepsy after SE, we used an AAV serotype 5 gene
transfer vector to express a GABR ␣1 cDNA in DG after SE. We
chose the novel approach of using the GABRA4 promoter to
express the ␣1 cDNA because GABRA4 promoter activity is upregulated in dentate after SE (Roberts et al., 2005). An AAV vector
containing either the ␣1 cDNA and eYFP reporter (AAV–␣1) or
the eYFP reporter only (AAV– eYFP) was injected into DG of
adult rats, and SE was induced 2 weeks later. SE was also induced
in two additional sets of control rats, saline-injected and uninjected naive. All of the rats were then continuously monitored for
spontaneous seizures and were killed 1, 2, or 4 weeks after SE.
AAV–␣1 injection selectively enhanced ␣1 subunit levels in
dentate after SE
Levels of 16 GABR subunit mRNAs (␣1–␣6, ␤1–␤3, ␥1–␥3, ␦, ␧,
␾, ␲) in microdissected DG were assessed by RNA amplification
and reverse Northern blotting. Rats injected with AAV–␣1
showed higher levels of ␣1 subunit by 2 weeks after SE (n ⫽ 5; p ⬍
0.02 by one-way ANOVA for effect of group; p ⬍ 0.01 for pairwise comparison by Bonferroni’s post hoc test) (Fig. 1 A). ␣1
mRNA and protein levels were similar between the control
groups (AAV– eYFP injections, n ⫽ 8; saline injections, n ⫽ 6; or
no injections, n ⫽ 3) and between the three time points at which
the control groups were analyzed (1, 2, or 4 weeks after SE; n ⫽ 8,
3, and 6, respectively), so all data were pooled as a single sham
control value (total n ⫽ 17). There were no significant differences
in mRNA levels between the AAV–␣1 and sham control groups
for any GABR subunit other than ␣1 (data not shown). To further quantify ␣1 mRNA changes, RT-PCR was performed. ␣1
mRNA levels were higher in DG from rats that were injected with
AAV–␣1 at 1 week (n ⫽ 6) and 2 weeks (n ⫽ 5) but not 4 weeks
(n ⫽ 4) after SE compared with sham controls ( p ⬍ 0.01 by
one-way ANOVA for effect of group; for pairwise comparison,
11344 • J. Neurosci., November 1, 2006 • 26(44):11342–11346
Raol et al. • Enhancing ␣1 Subunit Inhibits Epilepsy
Figure 1. GABR ␣1 subunit levels in the dentate gyrus after SE were higher in rats injected with AAV–␣1. a, ␣1 subunit mRNA (mean ⫾ SEM; normalized to cyclophilin) at different time points
after SE measured using RNA amplification. b, ␣1 mRNA levels measured with RT-PCR (normalized to cyclophilin). c, ␣1 subunit protein levels. Top, Representative Western blots demonstrating ␣1
subunit and ␤-actin protein levels in DG homogenates from sham control rat (c) and rats that were injected with AAV–␣1 and killed at 1, 2, and 4 weeks after SE. Bottom, Quantitation of protein
levels (mean ⫾ SEM) at different time points after SE (normalized to ␤-actin values and expressed as percentage of control values). d, Histogram represents eYFP mRNA levels (mean ⫾ SEM) in the
dentate gyrus of the rats that were killed after either 1 or 4 weeks after SE determined using RT-PCR. *p ⬍ 0.05; **p ⬍ 0.01; ***p ⬍ 0.001.
Figure 2. The SE characteristics were not different between AAV–␣1 and sham control rats.
a, Bar graphs (mean ⫾ SEM) shows the amount of pilocarpine required to induce SE was similar
between groups. b, The mean latency to onset of SE (in minutes) was not different between
sham and the AAV–␣1 group of rats. c, The histogram (mean ⫾ SEM) shows that the cumulative amount of diazepam required to stop seizures beginning 1 h after SE onset was the same
between groups.
p ⬍ 0.05 for 1 week and p ⬍ 0.01 for 2 week by Bonferroni’s post
hoc test) (Fig. 1 B). ␣1 subunit protein levels were also higher in
DG of the AAV–␣1-injected rats by 2 weeks after SE (n ⫽ 5; p ⬍
0.0001 by one-way ANOVA for effect of group; p ⬍ 0.001 by
Bonferroni’s post hoc test for pairwise comparison) (Fig. 1C) but
were similar to control by 4 weeks after SE (n ⫽ 4). To differentiate whether the decline in ␣1 subunit levels by 4 weeks after SE
was attributable to loss of transferred or endogenous ␣1, we assayed mRNA levels for eYFP (which is transcribed in tandem with
the ␣1 cDNA) in AAV-injected rats killed at 1 or 4 weeks after SE.
RT-PCR analysis demonstrated that mean eYFP mRNA levels
declined over sixfold in AAV-injected rats between 1 and 4 weeks
after SE (Fig. 1 D), suggesting that the decline in ␣1 subunit levels
by 4 weeks after SE most likely results from loss of ␣1 cDNA
expression from AAV rather than from changes in transcription
of the endogenous ␣1 gene or enhanced ␣1 turnover.
AAV–␣1 injection inhibited development of spontaneous
seizures after SE
All of the rats in both groups experienced SE after pilocarpine
injection, and there was no difference in any of the characteristics
of SE between the groups, including amount of pilocarpine required to induce SE (Fig. 2 A), latency to SE onset (Fig. 2 B), and
amount of diazepam required to stop seizures (Fig. 2C). All of the
rats were continuously video monitored from the time of SE until
Figure 3. Viral-mediated transfer of ␣1 subunit into DG decreased the risk of developing
spontaneous seizures after SE. a, The rate of spontaneous seizure development after SE per unit
time is lower in AAV–␣1 rats than in sham controls (Kaplan–Meier hazard function, p ⬍
0.0005). The cumulative hazard rate ( y-axis) is the summation of the probability per time unit
that a rat that has remained seizure free will develop seizures in a specific time interval. Censored cases, represented as ⫹, are cases in which rats had not developed first seizure by the end
of their follow-up period (total of 14 censored cases, all in the AAV–␣1 group, with follow-up
time ranging from 7 to 14 d). b, Length of time (mean ⫾ SEM) rats remained seizure free after
SE was threefold longer in AAV–␣1 rats compared with sham controls (***p ⬍ 0.0005).
the day they were killed to determine whether they developed
spontaneous behavioral seizures. AAV–␣1 injection significantly
increased the mean time to first spontaneous seizure after SE
( p ⬍ 0.0005 by Kaplan–Meier survival analysis; n ⫽ 23) (Fig.
3 A, B), and only 39% of AAV–␣1-injected rats (9 of 23) were
observed to develop spontaneous seizures compared with 100%
of rats receiving sham injections (n ⫽ 17). A subgroup of AAV–
␣1-injected rats (n ⫽ 7) were monitored using intracranial EEG
in addition to video monitoring to determine whether any electrographic seizure activity might be occurring in the absence of
behavioral seizures. No electrographic seizures were seen in the
absence of behavioral correlates, and all behavioral seizures were
associated with electrographic seizure activity. In AAV–␣1injected rats that went on to develop spontaneous seizures, the
severity and frequency of seizures did not differ from shaminjected rats.
In addition to the effects on seizures, elevation of ␣1 expression was also associated with a behavioral phenotype in a fraction
of the AAV–␣1-injected rats. Although most of the AAV–␣1injected rats behaved similarly to sham-injected rats, 30% were
observed to have abnormal behavior, including excessive seda-
Raol et al. • Enhancing ␣1 Subunit Inhibits Epilepsy
tion, anorexia, and weight loss that persisted for days to weeks
after SE, and was sometimes severe enough to require supplemental feedings and hydration to ensure survival. Formal testing
of memory and behavior was not performed. This observed behavioral effect was not seen after SE in any of the other groups
(including the AAV– eYFP control), suggesting that the effect
likely resulted from elevated ␣1 levels rather than the effects of
AAV injection or SE. Such effects are not surprising given that
␣1-containing receptors are the major form of GABRs mediating
sedation in the nervous system (Mohler et al., 2002).
Discussion
The current findings provide the first direct evidence that increasing the levels of a single GABAA receptor subunit, namely
the ␣1 subunit, in dentate gyrus can inhibit the development of
spontaneous seizures after SE. The current results are consistent
with a working model in which ␣1 subunits in DG are limiting
and their decrease in response to SE, in the background of increased ␣4, impairs inhibition in the hippocampus and contributes to the subsequent development of spontaneous seizures.
Some (Brooks-Kayal et al., 1998, 1999) but not all (Schwarzer et
al., 1997; Fritschy et al., 1999) previous studies of TLE have found
lower expression of ␣1 in the DG, however, and the importance
of ␣1 subunit changes for epileptogenesis has remained controversial. Although the present data do not prove a causal role for
␣1 changes in the pathogenesis of epilepsy, they strongly suggest
that ␣1 levels in DG can play a determining role in whether epilepsy develops after SE. It must be noted that the current shortterm study cannot determine whether enhanced ␣1 subunit expression is simply suppressing seizures (i.e., having an
anticonvulsant effect) or would permanently prevent development of epilepsy in some animals (i.e., an antiepileptogenic effect). Such a determination will require a much longer-term
study using a different viral construct that produces more persistent elevation of ␣1 subunit levels. Because SE is a known risk
factor for later development of epilepsy in humans (Cendes et al.,
1993), the current results suggest that such additional studies are
warranted to determine whether therapeutic strategies designed
to enhance GABR ␣1 subunit expression may be effective in reducing the risk of later epilepsy development in patients experiencing SE.
The specific cellular mechanisms by which the increased ␣1 is
exerting its effect on seizure development remains to be determined. Several potentially interesting future lines of investigation
are raised by our results. GABRs require multiple subunit proteins to be functional (typically 2␣, 2␤, and a ␥ or ␦). Thus, to
have an effect, the ␣1 expressed from the AAV vector must assemble with endogenous subunits, such as ␤ and ␥, and then be
trafficked to the cell membrane. Future studies to identify subunit partners of the viral ␣1 gene product within the cell and at
the cell surface after SE may provide insight into the mechanisms
of ␣1 gene transfer effects as well as assembly and trafficking of
␣1-containing GABRs. Physiological studies will be useful to assess the effect of ␣1 gene transfer on inhibitory function at both a
cellular and circuitry level. Finally, although in the current study,
we did not observe an obvious difference in hippocampal morphology between the AAV–␣1 and sham control groups using
routine light microscopy, more detailed studies including stereology and quantitative cell counting will be needed to be certain
that ␣1 gene transfer is not affecting SE-induced cell death or
synaptic reorganization.
Our results demonstrate that a vector containing the GABR
␣1 gene downstream of the GABRA4 promoter can be used to
J. Neurosci., November 1, 2006 • 26(44):11342–11346 • 11345
successfully increase ␣1 levels in DG. To our knowledge, this is
the first reported use of a condition-specific promoter upregulated in disease to drive enhanced expression of a gene that was
transferred by a somatic gene transfer vector into diseased cells.
Surprisingly, AAV transfer of the ␣1 transgene driven by the
GABRA4 promoter resulted in enhancement of ␣1 gene expression in DG for only the first 2 weeks after SE. The reduction of ␣1
expression at 4 weeks suggests that the GABRA4 promoter in
AAV vector is either silenced or transcriptionally downregulated.
Although gene silencing has been reported in the CNS for integrated retrovirus vectors (Prasad-Alur et al., 2002; Rosenquist et
al., 2005), it is less likely to alter AAV-mediated expression because AAV vector genomes are both episomal and associated with
high-molecular-weight DNA in the brain, and robust gene expression has been documented from AAV using various promoters for much longer than 4 weeks (Skorupa et al., 1999; Xu et al.,
2001; Passini et al., 2002). Our previous work strongly suggests
that BDNF and Egr3 (early growth response 3) are major regulators of enhanced ␣4 expression after SE (Roberts et al., 2005,
2006), so downregulation of these transcription factors could
explain our findings. However, the endogenous ␣4 subunit expression in AAV-injected animals did not vary at 1, 2, or 4weeks
after SE, as would be expected if Egr3 or BDNF were depleted or
downregulated, making this hypothesis unlikely. Additional
studies will be required to define the precise mechanism responsible for the decrease in expression of the ␣1 cDNA from AAV
over time.
Several previous studies have reported successful use of gene
transfer of inhibitory neuropeptides galanin (Haberman et al.,
2003; Lin et al., 2003; McCown, 2006) and neuropeptide Y
(Richichi et al., 2004) into the CNS to increase the threshold to
seizure induction by kainic acid or electrical kindling. However,
none of those studies examined the effects of gene transfer on
later development of spontaneous seizures or epilepsy after the
provoked seizure events. Another study in a genetic rat model in
which the aspartoacylase gene is deleted demonstrated that replacement of this gene reduced the spontaneous tonic convulsions that typically occur in this mutant but did not improve their
survival time (Seki et al., 2004). The current study provides the
first documentation of the successful use of viral gene transfer to
inhibit development of an acquired epilepsy. These findings suggest that strategies designed to modify abnormal gene expression
during the latent period between an initial precipitating injury
known to increase risk of epilepsy, such as status epilepticus or
severe head trauma, and the onset of spontaneous seizures may
have therapeutic potential for the prevention of epilepsy.
References
Brooks-Kayal AR, Shumate MD, Jin H, Rikhter TY, Coulter DA (1998) Selective changes in single cell GABAA receptor subunit expression and
function in temporal lobe epilepsy. Nat Med 4:1166 –1172.
Brooks-Kayal AR, Shumate MD, Jin H, Lin DD, Rikhter TY, Holloway KL,
Coulter DA (1999) Human neuronal ␥-aminobutyric acidA receptors:
coordinated subunit mRNA expression and functional correlates in individual dentate granule cells. J Neurosci 19:8312– 8318.
Buhl E, Otis T, Mody I (1996) Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model. Science 271:369 –373.
Cendes F, Andermann F, Dubeau F, Gloor P, Evans A, Jones-Gotman M,
Olivier A, Andermann E, Robitaille Y, Lopes-Cendes I (1993) Early
childhood prolonged febrile convulsions, atrophy and sclerosis of mesial
structures, and temporal lobe epilepsy: an MRI volumetric study. Neurology 43:1083–1087.
Fritschy JM, Kiener T, Bouilleret V, Loup F (1999) GABAergic neurons and
GABAA receptors in temporal lobe epilepsy. Neurochem Int 34:435– 445.
Gibbs III JW, Shumate MD, Coulter DA (1997) Differential epilepsy-
11346 • J. Neurosci., November 1, 2006 • 26(44):11342–11346
associated alterations in postsynaptic GABAA receptor function in dentate granule cells and CA1 neurons. J Neurophysiol 77:1924 –1938.
Haberman RP, Samulski RJ, McCown TJ (2003) Attenuation of seizures and
neuronal death by adeno-associated virus vector galanin expression and
secretion. Nat Med 9:1076 –1080.
Lin EJ, Richichi C, Young D, Baer K, Vezzani A, During MJ (2003) Recombinant AAV-mediated expression of galanin in rat hippocampus suppresses seizure development. Eur J Neurosci 18:2087–2092.
Macdonald RL, Olsen RW (1994) GABAA receptor channels. Annu Rev
Neurosci 17:569 – 602.
McCown TJ (2006) Adeno-associated virus-mediated expression and constitutive secretion of galanin suppresses limbic seizure activity in vivo.
Mol Ther 14:63– 68.
Mohler H, Fritschy JM, Rudolph U (2002) A new benzodiazepine pharmacology. J Pharmacol Exp Ther 300:2– 8.
Passini MA, Lee EB, Heuer GG, Wolfe JH (2002) Distribution of a lysosomal
enzyme in the adult brain by axonal transport and by cells of the rostral
migratory stream. J Neurosci 22:6437– 6446.
Passini MA, Watson DJ, Wolfe JH (2004) Gene delivery to the mouse brain
with adeno-associated virus. Methods Mol Biol 246:225–236.
Prasad-Alur RK, Foley B, Parente MK, Tobin DK, Heuer GG, Avadhani AN,
Pongubala J, Wolfe JH (2002) Modification of multiple transcriptional
regulatory elements in a Moloney murine leukemia virus gene transfer
vector circumvents silencing in fibroblast grafts and increases levels of
expression of the transferred enzyme. Gene Ther 9:1146 –1154.
Raol Y, Zhang G, Lund IV, Porter BE, Maronski MA and Brooks-Kayal AR
(2006) Increased GABA(A) receptor ␣1 subunit expression in hippocampal dentate gyrus after early-life status epilepticus. Epilepsia, in
press.
Richichi C, Lin EJ, Stefanin D, Colella D, Ravizza T, Grignaschi G, Veglianese
P, Sperk G, During MJ, Vezzani A (2004) Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus. J Neurosci 24:3051–3059.
Roberts DS, Raol Y, Bandyopadhyay S, Lund IV, Budreck EC, Passini MA,
Raol et al. • Enhancing ␣1 Subunit Inhibits Epilepsy
Wolfe JH, Brooks-Kayal AR, Russek SJ (2005) Egr3 stimulation of
GABRA4 promoter activity as a mechanism for seizure-induced upregulation of GABAA receptor alpha4 subunit expression. Proc Natl Acad
Sci USA 102:11894 –11899.
Roberts DS, Hu Y, Lund IV, Brooks-Kayal AR, Russek SJ (2006) BDNFinduced synthesis of early growth response factor 3 (EGR3) controls the
levels of type a GABA receptor alpha 4 subunits in hippocampal neurons.
J Biol Chem 281:29431–29435.
Rosenquist N, Jakobsson J, Lundberg C (2005) Inhibition of chromatin
condensation prevents transgene silencing in a neural progenitor cell line
transplanted to the rat brain. Cell Transplant 14:129 –138.
Schwarzer C, Tsunashima K, Wanzenbock C, Fuchs K, Sieghart W, Sperk G
(1997) GABAA receptor subunits in the rat hippocampus II: altered distribution in kainic acid-induced temporal lobe epilepsy. Neuroscience
80:1001–1017.
Seki T, Matsubayashi H, Amano T, Kitada K, Serikawa T, Sasa M, Sakai N
(2004) Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats. Neurochem Int 45:171–178.
Shumate MD, Lin DD, Gibbs III JW, Holloway KL, Coulter DA (1998)
GABAA receptor function in epileptic human dentate granule cells: comparison to epileptic and control rat. Epilepsy Res 32:114 –128.
Skorupa AF, Fisher KJ, Wilson JM, Parente MK, Wolfe JH (1999) Sustained
production of ␤-glucuronidase after somatic gene transfer results in disseminated distribution of the enzyme and reversal of distal lysosomal
storage lesions in the brains of mucopolysaccharidosis VII mice. Exp
Neurol 160:17–27.
Xu R, Janson CG, Mastakov M, Lawlor P, Young D, Mouravlev A, Fitzsimons
H, Choi KL, Ma H, Dragunow M, Leone P, Chen Q, Dicker B, During MJ
(2001) Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes. Gene Ther 8:1323–1332.
Zhang G, Raol Y, Hsu FC, Coulter DA, Brooks-Kayal AR (2004) Effects of
status epilepticus on hippocampal GABAA receptors are age-dependent.
Neuroscience 125:299 –303.